Composition comprising catalase, preparation method and use thereof and method for killing tumor cells

ABSTRACT

A composition comprising catalase, a preparation method and a use thereof, and a method for killing tumor cells are provided. The composition comprises a radionuclide labeled to a biomacromolecule, a soluble alginate and catalase. The composition can be injected into the tumor through an interventional treatment. A gel is formed when an alginate ion in the composition enters the tumor and encounters a calcium ion, such that the radionuclide and the catalase are uniformly confined in the tumor. The composition comprising catalase utilizes catalase to decompose dissolved oxygen generated from hydrogen peroxide in the tumor to advance the hypoxic state of the tumor cells, and the tumor cells are killed with radiation after the hypoxic state thereof has been advanced, and thus the invention has good prospects for applications in cancer therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a national stage application under 35 U.S.C.371 based on international patent application PCT/CN2018/091841, filedon Jun. 19, 2018 which claims the priority of Chinese Patent ApplicationNo. 201710467595.5 filed on Jun. 22, 2017, the disclosures of both ofwhich are incorporated in the present application by reference in theirentireties.

TECHNICAL FIELD

The present disclosure relates to the field of medical technology, andin particular to a composition for preparing an anti-tumor agent and usethereof, and specifically to a composition comprising catalase, apreparation method and use thereof and a method for killing tumor cells.

BACKGROUND

Radionuclides have different enrichment effects in different organtissues. For example, thyroid cells have a special affinity for iodide.After oral administration of a certain amount of iodine-131, the most ofthem can be absorbed by the thyroid gland. When iodine-131 decays intoxenon-131, β-rays (99%) and γ-rays (1%) are emitted. The former has aneffective range of 3.63 mm and an average range of 0.48 mm, which canselectively destroy the thyroid acinar epithelium without affectingadjacent tissues. However, the enrichment effects of radionuclides limittheir application to other tissues. In the prior art, minimally-invasiveimplantation of radioactive particles is one of the currently idealmethods. The radioactive material is placed in a micro-carrier, which isthen implanted at the lesion under the guidance of ultrasound or CT in aminimally-invasive surgery. The tumor cells will be killed by theradiation emitted upon particle decay. In this way, the carrierparticles will remain in the patient's body permanently, which will havelong-term effects on the patient's body. In addition, this implantationmethod has problems such as uneven distribution and poor coverage, whichis difficult to achieve a desired effect.

On the other hand, hypoxia is found in most of the human solid tumorsdue to the disordered growth of tumors and insufficient angiogenesis.The ability of tumor cells in hypoxic state to resist ionizing radiationis several times higher than that of normal oxygenated tumor cellsbecause the damage of DNA by oxygen radicals is reduced. Therefore, theradiation has a limited killing effect on hypoxic tumor cells, resultingin an in-apparent therapeutic effect of the existing radiationparticles.

SUMMARY

The present disclosure provides a composition comprising catalase, apreparation method and use thereof and a method for killing tumor cells.The composition can relieve the hypoxia in tumor cells, increase theuniformity of the distribution of radioactive particles and the abilityof radiative rays to kill tumor cells without affecting the surroundingnormal tissues.

To achieve at least one of the above purposes of the present disclosure,the present disclosure may employ the following technical solutions:

The disclosure provides a catalase-comprising composition, whichcomprises catalase, a soluble alginate, and a radionuclide labeled on abio-macromolecule.

A gel is formed when the alginate ions in the composition enter into thetumor and encounter calcium ions, and adheres to the inside of thetumor, such that the radionuclide-labeled bio-macromolecules andcatalase are uniformly confined within the tumor. The catalase candecompose hydrogen peroxide in the tumor to produce dissolved oxygen,thereby relieving the hypoxia in tumor cells. In addition, theradioactive rays can ionize the dissolved oxygen to generate oxygen freeradicals which can kill tumor cells, thereby greatly enhancing thekilling effect of radioactive rays on tumor cells.

On the other hand, the soluble alginate, catalase andradionuclide-labeled bio-macromolecules can form a network-likeuniformly-distributed gel structure according to the internal structureof the tumor. The catalase and radionuclide-labeled bio-macromoleculesare confined in the gel structure formed from the alginate due to theirsuitable physical structure without being carried by the blood to thepatient's normal tissue area, thereby reducing the overall dosage ofradionuclides while ensuring the intensity of radiative rays in thelesion area. In addition, the alginate gradually degrades within a fewweeks in organisms, leaving no particles.

Optionally, in the composition comprising catalase, the soluble alginateincludes at least one selected from the group consisting of sodiumalginate, potassium alginate, ammonium alginate and propylene glycolalginate. After the soluble alginate is dissolved into a solution, thealginate ions in the solution will crosslink with calcium ions to form athree-dimensional network-like gel. Sodium alginate is a naturalpolysaccharide which has the stability, solubility and viscosityrequired for adjuvants of pharmaceutical preparations.

Optionally, in the composition comprising catalase, the soluble alginateis potassium alginate, wherein potassium ions promote the contraction ofblood vessels and reduce the fluidity of the injected fluid in tumors.

Optionally, in the composition comprising catalase, the soluble alginateis a mixture of sodium alginate and potassium alginate. The proportionin the mixture can be adjusted according to the different conditions ofthe blood vessels in the tumor, thereby adjusting the contraction degreeof the blood vessels and adjusting the distribution state of thecomposition in the blood vessels.

Optionally, in the composition comprising catalase, the radionuclideincludes at least one selected from the group consisting of iodine-131,iodine-125, phosphorus-32, yttrium-90, gallium-67, indium-111,thallium-201, palladium-203, bismuth-213, actinium-225, lutecium-177,rhenium-186, palladium-212 and rhenium-188. In the lesion area,radionuclides kill and inhibit tumor cells by utilizing theirradioactivity.

Optionally, in the composition comprising catalase, the radionuclide isiodine-131 or iodine-125.

Optionally, in the composition comprising catalase, theradionuclide-labeled bio-macromolecule is at least one selected from thegroup consisting of a protein, a nucleic acid and a polysaccharide. Thebio-macromolecules of the present disclosure are bio-macromolecules wellknown in the art and are useful as carriers for radionuclides, includingenzymes and serum proteins.

Optionally, in the composition comprising catalase, the radionuclide islabeled on the catalase. The uniformity of the relative distancedistribution between the radiation and the dissolved oxygen generated bythe catalase can be further defined by labeling a radionuclide on thecatalase, such that no non-uniform spatial distribution is occurred as aresult of the differences in molecular weight. Thereby, the generationprobability of oxygen free radicals and their killing effects on tumorcells are increased.

Optionally, in the composition comprising catalase, the concentration ofthe catalase is 5 mg/mL-10 mg/mL, the concentration of the alginate is 5mg/mL-10 mg/mL and the dose of the radionuclide is 200 Ci/mL-500 Ci/mL.Alginate at a concentration of 5 mg/mL to 10 mg/mL has a good gelationability, which allows the composition to spread well within the tumor.If the alginate has a concentration of less than 5 mg/mL, it's gelationability is insufficient, and the composition is likely to flow out ofthe tumor. If the alginate has a concentration of more than 10 mg/mL, itgels too fast, causing the composition to stay in a certain part of thetumor. Both cases affect the efficacy of the composition.

Optionally, in the composition comprising catalase, the compositionfurther comprises an immune-stimulating agent.

Optionally, in the composition comprising catalase, theimmune-stimulating agent includes at least one selected from the groupconsisting of CpG oligodeoxynucleotide which is an immunologic adjuvant,R837, TLR7 agonists, TLR8 agonists, NLR agonists, STING agonists, MPLA(Monophosphoryl Lipid A), LPS, PGNs, R848, G100, SD-101, MGN1703,CMP-001, FLA, polyU, poly(dT), CL307, CL264, CL097, CL075, MEDI9197,MEDI5083, hypoxanthine and MDP. Cytokine includes IL-1, IL-1α, IL-1β,IL-2, IL-2 superkine, IL-6, IL-7, IL-9, AM0010, IL-12, IL-15, IL-15superagonist, ALT-803, NIZ985, IL-16, IL-18, IL-21, denenicokin, IL-12superagonist antibody, IFN-α, IFN-β, IFN-γ, TNF-α, GM-GSF, RG7461,RG7813, M9241, etc. The immune-stimulating agent may be used incombination with antibodies against immune checkpoint blockers such asan antibody against cytotoxic T lymphocyte-associated antigen-4(Anti-CTLA-4), Anti-PD-1, Anti-PD-L1, etc. and costimulatory signalmolecules such as Anti-TIM3, Anti-OX40, Anti-GITR, Anti-LAG-3,Anti-CD137, Anti-CD27, Anti-CD28, Anti-CD28H, Anti-CD39, Anti-CD40,Anti-CD47, Anti-CD48, Anti-CD70, Anti-CD73, Anti-CD96, Anti-CD160,Anti-CD200, Anti-CD244, etc. to produce suitable amount of antigens forimmune therapy. The addition of an immunologic adjuvant to thecomposition results in a better production of tumor-associated antigens.Then the use of an antibody against the immune checkpoint blocker forimmune response modulation facilitates to kill distal tumors ormetastases by utilizing the autoimmune. Thus, the distal tumors ormetastases are treated while treating in situ tumors.

The present disclosure provides a method for preparing a compositioncomprising catalase, comprising mixing an alginate solution, aradionuclide and catalase.

Optically, the ratio of the amount of the alginate, the catalase, andthe radionuclide is 5 mg-10 mg: 5 mg-10 mg: 200 Ci-500 Ci.

Optically, the alginate solution, radionuclide and catalase are mixed bya method comprising mixing an alginate solution withradionuclide-labeled catalase.

Further, as an implementation, the radionuclide-labeled catalase isprepared by a method comprising mixing a radionuclide, an oxidizingagent and a dispersion medium, and mixing the obtained mixture with abio-macromolecule such as catalase and serum proteins, then shaking,standing, washing and separating. The radionuclide is mixed with theoxidizing agent for oxidation, which gives the radionuclide a strongerelectrophilic substitution capability. The oxidizing agent includeschloramine-T, and the dispersion medium includes at least one selectedfrom the group consisting of physiological saline and phosphate bufferedsaline (PBS).

As another implementation, the radionuclide-labeled catalase is preparedby a method comprising mixing catalase with a chelating agent and thendialyzing to obtain chelated catalase; mixing a radionuclide with thechelated catalase and a reducing agent, followed by ultrafiltrationpurification. A chelating agent is used in this method such that achelating agent capable of chelating radioactivity is present on thecatalase, i.e., the catalase is modified on the surface with a chelatingagent capable of labeling a radionuclide. The radionuclide is thenchelated with the chelating agent by using a reducing agent to obtainradionuclide-labeled catalase. In the present disclosure, the chelatingagent includes DTPA-based and DOTA-based chelating agents, and thereducing agent includes sodium borohydride or potassium borohydride.

The present disclosure provides the use of the above-describedcomposition comprising catalase for the preparation of ananti-neoplastic agent.

Optically, the use comprises use of the composition in combination withat least one selected from the group consisting of an antibody againstcytotoxic T-lymphocyte-associated antigen-4, anti-PD-1 and anti-PD-L1.Suitable amount of antigens is produced for immune therapy, and distaltumors or metastases are killed by utilizing the autoimmune. Thus, thedistal tumors or metastases are treated while treating in situ tumors.

The present disclosure also provides a method for killing tumor cells.The above composition is injected into a tumor by means ofinterventional treatment. A network-like cross-linked gel structure isformed according to the internal structure of the tumor when thealginate ions in the composition enter into the tumor and encountercalcium ions, and adheres to the inside of the tumor, such that theradionuclides labeled on bio-macromolecules and catalase are uniformlycoated and confined within the tumor. The catalase can decomposehydrogen peroxide in the tumor to produce dissolved oxygen, therebyrelieving the hypoxia in tumor cells. The rays generated by theradionuclides can kill tumor cells in which the hypoxia has beenrelieved.

Optionally, prior to interventional treatment, the calcium concentrationin blood or in tumors and surrounding tissue regions is adjusted.

Advantages of the present disclosure include:

In the present disclosure, a gel is formed from alginate ions andcalcium ions, and adheres to the inside of the tumor, such that theradionuclide-labeled bio-macromolecules and catalase are uniformlyconfined within the tumor. The catalase can decompose hydrogen peroxidein the tumor to produce dissolved oxygen, thereby relieving the hypoxiain tumor cells. In addition, the radioactive rays can ionize thedissolved oxygen to generate oxygen free radicals to kill tumor cells,thereby greatly enhancing the killing effect of radioactive rays ontumor cells. Thereby, the problem of radiation resistance of hypoxictumor cells existing in the prior art is solved.

The network-like uniformly-distributed gel structure formed from thealginate ions and calcium ions defines the uniformity of the relativedistance distribution between the radiative rays and the dissolvedoxygen generated by the catalase, thereby improving the problems in theprior art that the non-uniform distribution and poor coverage ofradionuclides that has been placed in carrier particles in advance willoccur after being implanted into the lesion area.

In addition, the composition may be used in combination with an antibodyagainst cytotoxic T lymphocyte antigen-4 (CTLA-4) to treat both in situtumors and distal or metastatic tumors because antigens may be generatedwhen killing tumor cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is further described below in conjunction withthe accompanying drawings and embodiments:

FIG. 1 is a graph showing the imaging to monitor the movement ofradionuclides in mouse tumors within 48 h in Test Example 1;

FIG. 2 is a graph showing the photoacoustic imaging to monitor theoxygen concentration in the blood of mouse tumors;

FIG. 3 is a graph showing the tumor growth curve in Test Example 3;

FIG. 4 is a graph showing the tumor growth curve in Test Example 4;

FIG. 5 is a graph showing the mouse survival curve in Test Example 4;

FIG. 6 is a graph showing the in situ tumor growth curve;

FIG. 7 is a graph showing the distal tumor growth curve;

FIG. 8 is a graph showing the content of immune-related cytokines inmice;

FIG. 9 is a graph showing sections showing the permeation behavior ofsodium alginate of different concentrations in tumors;

FIG. 10 is a graph showing the tumor growth curve in Test Example 8;

FIG. 11 is a graph showing the tumor growth curve in Test Example 9.

DETAILED DESCRIPTION

The technical solutions in the embodiments of the present disclosurewill be clearly and completely described below in order to clarify theobjects of the examples, the technical solutions and the advantages ofthe present disclosure. The examples which are not indicated withspecific conditions are carried out according to the conventionalconditions or the conditions recommended by the manufacturer. Thereagents or instruments for use, which are not indicated withmanufacturers, are conventional products that are commerciallyavailable.

The present disclosure is described in detail below with reference tothe examples and the accompanying drawings. However, the followingexamples are not to be considered as limitations to the scope of thepresent disclosure.

Example 1

Preparation of a sodium alginate solution: 50 mg of sodium alginate wasweighed and dissolved in 10 ml of deionized water, and fully shaken tosufficiently dissolve the sodium alginate to obtain a 5 mg/ml sodiumalginate solution.

Preparation of iodine-125-labeled catalase: 550 μl of iodine-125 stocksolution and 100 μl of 0.1 mg/ml chloramine-T were mixed, and 500 μl ofdispersion medium was added, then the mixture of iodine-125 andchloramine-T was added to 500 μl of 5 mg/ml catalase, shaken and mixedon a vortex shaker for 5 minutes, then allowed to stand at roomtemperature for 15 minutes. 15 minutes later, the mixture wastransferred into a 15 ml ultrafiltration tube and centrifuged at 4000rpm for 15 minutes, washed 3 times with dispersion medium, then thesupernatant was taken out and measured for activity with an activitymeter.

The sodium alginate solution and the iodine-125-labeled catalase wereuniformly mixed to obtain a composition comprising catalase, wherein theconcentration of catalase was 5 mg/mL, the concentration of sodiumalginate was 5 mg/mL and the dose of radionuclide was 200 Ci/mL.

Example 2

Preparation of a sodium alginate solution: 75 mg of sodium alginate wasweighed and dissolved in 10 ml of deionized water, and fully shaken tosufficiently dissolve the sodium alginate to obtain a 7.5 mg/ml sodiumalginate solution.

Preparation of iodine-125-labeled catalase: 550 μl of iodine-125 stocksolution and 100 μl of 0.1 mg/ml chloramine-T were mixed, and 500 μl ofdispersion medium was added, then the mixture of iodine-125 andchloramine-T was added to 500 μl of 5 mg/ml catalase, shaken and mixedon a vortex shaker for 5 minutes, then allowed to stand at roomtemperature for 15 minutes. 15 minutes later, the mixture wastransferred into a 15 ml ultrafiltration tube and centrifuged at 4000rpm for 15 minutes, washed 3 times with dispersion medium, then thesupernatant was taken out and measured for activity with an activitymeter.

The sodium alginate solution and the iodine-125-labeled catalase wereuniformly mixed to obtain a composition comprising catalase, wherein theconcentration of catalase was 7.5 mg/mL, the concentration of sodiumalginate was 7.5 mg/mL and the dose of radionuclide was 350 Ci/mL.

Example 3

Preparation of a sodium alginate solution: 100 mg of sodium alginate wasweighed and dissolved in 10 ml of deionized water, and fully shaken tosufficiently dissolve the sodium alginate to obtain a 10 mg/ml sodiumalginate solution.

Preparation of iodine-125-labeled catalase: 550 μl of iodine-125 stocksolution and 100 μl of 0.1 mg/ml chloramine-T were mixed, and 500 μl ofdispersion medium was added, then the mixture of iodine-125 andchloramine-T was added to 500 μl of 5 mg/ml catalase, shaken and mixedon a vortex shaker for 5 minutes, then allowed to stand at roomtemperature for 15 minutes. 15 minutes later, the mixture wastransferred into a 15 ml ultrafiltration tube and centrifuged at 4000rpm for 15 minutes, washed 3 times with dispersion medium, then thesupernatant was taken out and measured for activity with an activitymeter.

The sodium alginate solution and the iodine-125-labeled catalase wereuniformly mixed to obtain a composition comprising catalase, wherein theconcentration of catalase was 10 mg/mL, the concentration of potassiumalginate was 10 mg/mL and the dose of radionuclide was 500 Ci/mL.

Example 4

Preparation of a potassium alginate solution: 550 mg of potassiumalginate was weighed and dissolved in 10 ml of deionized water, andfully shaken to sufficiently dissolve the potassium alginate to obtain a55 mg/ml potassium alginate solution.

Preparation of iodine-125-labeled catalase: 550 μl of iodine-125 stocksolution and 100 μl of 0.1 mg/ml chloramine-T were mixed, and 500 μl ofdispersion medium was added, then the mixture of iodine-125 andchloramine-T was added to 500 μl of 5 mg/ml catalase, shaken and mixedon a vortex shaker for 5 minutes, then allowed to stand at roomtemperature for 15 minutes. 15 minutes later, the mixture wastransferred into a 15 ml ultrafiltration tube and centrifuged at 4000rpm for 15 minutes, washed 3 times with dispersion medium, then thesupernatant was taken out and measured for activity with an activitymeter.

The potassium alginate solution and the iodine-125-labeled catalase wereuniformly mixed to obtain a composition comprising catalase, wherein theconcentration of catalase was 5 mg/mL, the concentration of potassiumalginate was 55 mg/mL and the dose of radionuclide was 200 Ci/m L.

Example 5

Preparation of a sodium alginate solution: 75 mg of sodium alginate wasweighed and dissolved in 10 ml of deionized water, and fully shaken tosufficiently dissolve the sodium alginate to obtain a 7.5 mg/ml sodiumalginate solution.

Preparation of iodine-131-labeled catalase: 50 μl of iodine-131 stocksolution and 100 μl of 0.1 mg/ml chloramine-T were mixed, and 500 μl ofPBS or physiological saline was added, then the mixture of iodine-131and chloramine-T was added to 500 μl of 5 mg/ml catalase, shaken andmixed on a vortex shaker for 5 minutes, then allowed to stand at roomtemperature for 15 minutes. 15 minutes later, the mixture wastransferred into a 15 ml ultrafiltration tube and centrifuged at 4000rpm for 15 minutes, washed 3 times with dispersion medium, then thesupernatant was taken out and measured for activity with an activitymeter.

The sodium alginate solution and the iodine-131-labeled catalase wereuniformly mixed to obtain a composition comprising catalase, wherein theconcentration of catalase was 5 mg/mL, the concentration of potassiumalginate was 7.5 mg/mL and the dose of radionuclide was 200 Ci/mL.

Example 6

Preparation of a potassium alginate solution: 100 mg of potassiumalginate was weighed and dissolved in 10 ml of deionized water, andfully shaken to sufficiently dissolve the potassium alginate to obtain a10 mg/ml potassium alginate solution.

Preparation of iodine-131-labeled catalase: 50 μl of iodine-131 stocksolution and 100 μl of 0.1 mg/ml chloramine-T were mixed, and 500 μl ofPBS or physiological saline was added, then the mixture of iodine-131and chloramine-T was added to 500 μl of 5 mg/ml catalase, shaken andmixed on a vortex shaker for 5 minutes, then allowed to stand at roomtemperature for 15 minutes. 15 minutes later, the mixture wastransferred into a 15 ml ultrafiltration tube and centrifuged at 4000rpm for 15 minutes, washed 3 times with dispersion medium, then thesupernatant was taken out and measured for activity with an activitymeter.

The potassium alginate solution and the iodine-131-labeled catalase wereuniformly mixed to obtain a composition comprising catalase, wherein theconcentration of catalase was 10 mg/mL, the concentration of potassiumalginate was 10 mg/mL and the dose of radionuclide was 500 Ci/m L.

Example 7

Preparation of a sodium alginate solution: 50 mg of sodium alginate wasweighed and dissolved in 10 ml of deionized water, and fully shaken tosufficiently dissolve the sodium alginate to obtain a 5 mg/ml sodiumalginate solution.

1 ml of catalase (50 mg/ml) was mixed with 1 mL of NHS-DTPA (1 μg/ml)for one hour, and then dialyzed against water for one day by using adialysis bag with a molecular weight cut-off of 3000 to obtainDTPA-catalase.

Preparation of technetium-99-labeled catalase: Technetium-99 (200 μci)was mixed and shaken with DTPA-catalase and 1 μg of sodium borohydridefor 10 minutes, and purified by ultrafiltration to obtaintechnetium-99-labeled catalase.

The sodium alginate solution and the technetium-99-labeled catalase wereuniformly mixed to obtain a composition comprising catalase, wherein theconcentration of catalase was 5 mg/mL, the concentration of sodiumalginate was 5 mg/mL and the dose of radionuclide was 200 Ci/mL.

Example 8

Preparation of a sodium alginate solution: 100 mg of sodium alginate wasweighed and dissolved in 10 ml of deionized water, and fully shaken tosufficiently dissolve the sodium alginate to obtain a 10 mg/ml sodiumalginate solution.

2 ml of catalase (50 mg/ml) was mixed with 1 mL of NHS-DTPA (1 μg/ml)for one hour, and then dialyzed against water for one day by using adialysis bag with a molecular weight cut-off of 3000 to obtainDTPA-catalase.

Preparation of rhenium-188-labeled catalase: Rhenium-188 (500 μci) wasmixed and shaken with DTPA-catalase and 1 μg of sodium borohydride for10 minutes, and purified by ultrafiltration to obtainrhenium-188-labeled catalase.

The sodium alginate solution and the rhenium-188-labeled catalase wereuniformly mixed to obtain a composition comprising catalase, wherein theconcentration of catalase was 10 mg/mL, the concentration of sodiumalginate was 10 mg/mL and the dose of radionuclide was 500 Ci/m L.

Comparative Example 1

Preparation of a sodium alginate solution: 50 mg of sodium alginate wasweighed and dissolved in 10 ml of deionized water, and fully shaken tosufficiently dissolve the sodium alginate to obtain a 5 mg/ml sodiumalginate solution.

Preparation of sodium alginate-combined ionized iodine-131: 50 μL of theiodine-131 stock solution was mixed with 5 mg/ml sodium alginatesolution.

Comparative Example 2

Preparation of a sodium alginate solution: 50 mg of sodium alginate wasweighed and dissolved in 10 ml of deionized water, and fully shaken tosufficiently dissolve the sodium alginate to obtain a 5 mg/ml sodiumalginate solution.

Preparation of iodine-131-labeled human serum albumin: 50 μl ofiodine-131 stock solution and 100 μl of 0.1 mg/ml chloramine-T weremixed, and 500 μl of dispersion medium was added, then the mixture ofiodine-131 and chloramine-T was added to 500 μl of 5 mg/ml human serumalbumin, shaken and mixed on a vortex shaker for 5 minutes, then allowedto stand at room temperature for 15 minutes. 15 minutes later, themixture was transferred into a 15 ml ultrafiltration tube andcentrifuged at 4000 rpm for 15 minutes, washed 3 times with dispersionmedium, then the supernatant was taken out and measured for activitywith an activity meter.

The sodium alginate solution, iodine-131-labeled human serum albumin andcatalase solution were uniformly mixed to obtain a compositioncomprising catalase, wherein the concentration of catalase was 5 mg/mL,the concentration of alginate was 5 mg/mL and the dose of radionuclidewas 200 Ci/mL.

Test Example 1

FIG. 1 is a graph showing the imaging to monitor the movement ofradionuclides in mouse tumors within 48 h. A series of experiments wereperformed on mice to verify that the alginate gel can confine theradionuclide labeled on the bio-macromolecule within the tumor.

Wherein the concentration of sodium alginate was 5 mg/mL, theconcentration of catalase was 5 mg/mL and the dose of iodine 131 was 200Ci/mL. The dispersion medium was PBS solution, and 50 μl was injectedinto each mouse. 6 breast cancer-bearing mice were divided into 6 groupsand intratumorally injected with:

A: ionized iodine-131;

B: iodine-131-labeled human serum albumin;

C: the iodine-131-labeled catalase from Example 6;

D: the sodium alginate-combined ionized iodine-131 as provided inComparative example 1

E: the sodium alginate-combined iodine-131-labeled human serum albuminas provided in Comparative example 2

F: the sodium alginate-combined iodine-131-labeled catalase as providedin Example 6.

The movement of the injected composition was observed with a SmallAnimal Radionuclide Imager at 0, 4, 12, 24 and 48 h after theintratumoral injection. The movement of sodium alginate and iodine131-labeled catalase was monitored in real time by using the radiativerays emitted by iodine 131 nuclide after the direct injection into mousetumors. As can be seen from FIG. 1, in the control group without sodiumalginate, the composition had substantially leaked out of the tumor andbeen metabolized from the mouse through an intravenous route at 24 hoursafter injection. However, the sodium alginate effectively retained theiodine-131-labeled bio-macromolecules within the tumor. Thus, it wasindicated that a gel was formed when the sodium alginate was injectedinto the tumor and encountered trace amounts of calcium irons in thetumor, and the gel effectively coated the bio-macromolecules andconfined them within the tumor.

Test Example 2

FIG. 2 is a graph showing the photoacoustic imaging to monitor theoxygen concentration in the blood of mouse tumors. The upper legend is alegend of photoacoustic imaging, and the lower legend is a legend ofultrasound imaging. The ability of the composition comprising catalaseto relieve hypoxia in the mouse 4T1 tumor models was further verified. 4breast cancer-bearing mice were intratumorally injected with human serumalbumin, catalase, sodium alginate-combined human serum albumin, andsodium alginate-combined catalase respectively. Photoacoustic was usedto monitor the oxygen concentration in the blood of tumors. The graypart in the figure showed the shape of the tumor detected by ultrasoundimaging, and the white part showed the distribution of dissolved oxygen.It can be seen from FIG. 2 that the combination of sodium alginate andcatalase relieved tumor hypoxia for a long time. Due to the presence ofthe sodium alginate gel, the catalase was well fixed within the tumor,thereby obtaining a long-term hypoxia-relieving effect.

Test Example 3

The breast cancer-bearing mice were divided into 2 groups with 5 mice ineach group and subjected to parallel experimental treatments. The 2groups of mice were intratumorally injected with physiological salineand a composite of catalase and sodium alginate respectively. After theintratumoral injection, the length and width of the tumor were measuredwith a vernier caliper every two days, and the volume of the tumor wascalculated as (length×(width×width))÷2. FIG. 3 is a graph showing tumorgrowth curves and mouse survival curves. It can be seen that thecomposite of catalase and sodium alginate had no killing effect on tumortissues at the test concentration.

Test Example 4

FIG. 4 and FIG. 5 were graphs showing the tumor growth curves of miceand the survival curves of mice, respectively.

The breast cancer-bearing mice were divided into 5 groups with 5 mice ineach group and subjected to parallel experimental treatments. The 5groups of mice were intratumorally injected with physiological saline,iodine-131-labeled human serum albumin, iodine-131-labeled catalase, thesodium alginate-combined iodine-131-labeled human serum albumin asprovided in Comparative example 2 and the sodium alginate-combinediodine-131-labeled catalase as provided in Example 6 respectively. Afterthe intratumoral injection, the length and width of the tumor weremeasured with a vernier caliper every two days, and the volume of thetumor was calculated as (length×(width×width))÷2. It can be seen fromthe tumor growth curves and the mouse survival curves (FIG. 3 and FIG.4) that the system enhanced the therapeutic ability of iodine-131,exhibiting a good therapeutic effect. In addition, the mice in theexperimental group using a combination of sodium alginate and catalasesurvived for the longest time. Wherein, “control” represented a controlgroup wherein physiological saline alone was injected and no therapeuticeffect was shown; “131I-HAS” represented human serum albumin labeledwith iodine 131 alone, wherein only the therapeutic effect of iodine 131was shown; “131I-Cat” represented iodine 131-labeled catalase, whereinthe therapeutic effect of iodine 131 after hypoxia relief was shown;“131I-HSA/ALG” represented sodium alginate-combined iodine131-labeledhuman serum albumin; and finally, “131I-Cat/ALG” represented sodiumalginate-combined iodine 131-labeled catalase.

Test Example 5

FIG. 6 and FIG. 7 were graphs showing the in situ tumor growth curvesand distal tumor growth curves of mice, respectively.

The tumor-bearing mice inoculated with mouse colon cancer on both sidesof the buttocks were divided into 5 groups with 5 mice in each group,and subjected to treatment involving combined immunotherapy. The 5groups of mice were intratumorally injected respectively with

A physiological saline;

B the sodium alginate-combined iodine 131-labeled catalase as providedin Example 6;

C the sodium alginate-combined iodine 131-labeled catalase as providedin Example 6 in combination with an antibody against cytotoxicT-lymphocyte-associated antigen-4. Specifically, the sodiumalginate-mixed iodine 131-labeled catalase was injected into the in situtumor, and the antibody against cytotoxic T lymphocyte-associatedantigen-4 was intravenously administered into the body.

D a composition of the sodium alginate-combined iodine 131-labeledcatalase as provided in Example 6 and CpG oligonucleotide. At the timeof injection, the sodium alginate-combined iodine 131-labeled catalasewas directly mixed with the CpG oligonucleotide composition and injectedin situ into the tumor.

E a composition of the sodium alginate-combined iodine 131-labeledcatalase as provided in Example 6 and CpG oligonucleotide in combinationwith an antibody against cytotoxic T-lymphocyte-associated antigen-4.The injection was performed as follows: the sodium alginate-combinediodine 131-labeled catalase was directly mixed with the CpGoligonucleotide composition and injected in situ into the tumor, and theantibody against cytotoxic T lymphocyte-associated antigen-4 wasintravenously administered into the body.

After the intratumoral injection into the in situ tumor, the length andwidth of the in situ tumor and the distal tumor were measured with avernier caliper every two days, and the volume of the tumor wascalculated as (length×(width×width))÷2. As can be seen from the in situtumor growth curves and the distal tumor growth curves (FIG. 5 and FIG.6), an internal radiotherapy realized by the sodium alginate-combinediodine-131-labeled catalase effectively and almost completely eliminatedin situ tumors, which resulted in the generation of related antigens.Thus, when the composition was used in combination with animmune-stimulating nucleotide and an antibody against cytotoxic Tlymphocyte-associated antigen-4, the distal tumors were effectivelyinhibited, thereby achieving a completed treatment of cancer.

Test Example 6

After 20 days of treatment, blood was collected from the mouse eyeballsand the mouse tumors were collected by dissection. The collected bloodwas allowed to stand at room temperature for 1.5 h and then centrifugedat 3000 rpm for 20 minutes to separate the serum. Immune-relatedcytokines in serum and tumors were measured by enzyme-linkedimmunosorbent assay.

As can be seen from FIG. 8, compared with the four control groups A, B,C and D, because the mice in the key experimental group wereintratumorally injected with a composition of sodium alginate,iodine-131-labeled catalase and an immunologic adjuvant oligonucleotideand intravenously administered with anti-cytotoxic Tlymphocyte-associated antigen 4, the in situ tumors were eliminated bythe internal radiotherapy and a large number of related antigens wasgenerated. In addition, related immune factors such as cytotoxic T cellswere increased and regulatory T cells were reduced in mice as a resultof the combination with an immunologic adjuvant and animmune-stimulating agent, indicating that lymphocytes with tumor-killingproperties were increased and lymphocytes that protected tumors werereduced in mice. Further detection of cytokines in distal tumorsrevealed that gamma interferon was increased accordingly which alsoindicated that tumor-killing lymphocytes within tumors were increased,and that tumor necrosis factor alpha was increased which indicated thatthe distal tumors were severely necrotic by lymphocyte challenge. Thisseries of phenomena indicated that the combination use of an immunologicadjuvant and an immune-stimulating agent significantly increased thetumor-killing lymphocytes in mice after the removal of in situ tumorswith internal radiotherapy, and these lymphocytes recognized andattacked distal tumor cells in mice and made them necrotic, whichconvincingly explained the good effect of internal radiotherapy combinedwith immunotherapy.

Test Example 7

The penetration behavior of different concentrations of sodium alginatein mouse tumors were further investigated. The catalase was labelledwith fluorescein dye as follows: 10 μl of fluorescein dye was added to 1ml of 5 mg/ml catalase, then 10 mg of1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added,stirred for 12 hours, then 1.1 ml of the reaction solution wastransferred to dialysis bag having a molecular weight cut-off of 14800and dialyzed against water for two days, and water was changed every 12hours. After dialysis, fluorescein dye-labeled catalase was obtained.The catalase was mixed with different concentrations of sodium alginate(1, 5, 10, and 20 mg/ml), and injected intratumorally into tumors oftumor-bearing mice. Tumors were collected at 4 hours and 72 hours,respectively, and tumor sections were stained. As can be seen from FIG.9, the sodium alginate at a concentration of 5 mg/ml had a suitablegelation ability and allowed the composition to spread well within thetumor, compared to that at a concentration of less than 5 mg/ml whichhad an insufficient gelation ability and caused the composition to flowout of the tumor at 72 hours, and that at a concentration of more than10 mg/ml which gelled too fast and caused the composition to stay in acertain part of the tumor. In summary, it can be concluded that 5-10mg/ml was the optimum concentration of sodium alginate. Theconcentration of calcium ions in mice is in the range of1.5*10-3˜1.8*10-3M, and the concentration in human body is in the rangeof 1.5*10-3˜1.8*10-3M too, so the concentration of sodium alginate forthe human body is also 5 to 10 mg/ml.

Test Example 8

FIG. 10 was a graph showing the tumor growth curve of mice.

The breast cancer-bearing mice were divided into 3 groups with 5 mice ineach group and subjected to parallel experimental treatments. The 3groups of mice were intratumorally injected with a composition ofcatalase labeled by different doses of iodine 131 and sodium alginate,wherein the dose of iodine 131 was 10, 20 and 50 microcuriesrespectively. After the intratumoral injection, the length and width ofthe tumor were measured with a vernier caliper every two days, and thevolume of the tumor was calculated as (length×(width×width))÷2. It canbe seen from the tumor growth curve (FIG. 10) that the tumortransplantation effect of the composition was gradually relieved as thedose of iodine 131 increased, and 50 μCi of iodine 131 eliminated thetumors in mice completely.

Test Example 9

FIG. 11 was a graph showing the tumor growth curve of mice.

The breast cancer-bearing mice were divided into 3 groups with 5 mice ineach group and subjected to parallel experimental treatments. The 3groups of mice were intratumorally injected with a composition ofcatalase labeled by different doses of iodine 125 and sodium alginate,wherein the dose of iodine 125 was 10, 20 and 50 microcuriesrespectively. After the intratumoral injection, the length and width ofthe tumor were measured with a vernier caliper every two days, and thevolume of the tumor was calculated as (length×(width×width))÷2. It canbe seen from the tumor growth curve (FIG. 11) that the tumortransplantation effect of the composition was gradually increased as thedose of iodine 125 increased, and 50 μCi of iodine 125 had the besttherapeutic effect.

In addition, it should be understood that although the description isdescribed in terms of the embodiments, not each embodiment includes onlyone independent technical solution. The description of the specificationis merely for the sake of clarity, and those skilled in the art shouldregard the specification as a whole. The technical solutions in therespective examples may also be combined appropriately to form otherembodiments that can be understood by those skilled in the art. Allother examples obtained by those general skilled in the art under thepremise of no creative work, on the basis of examples of the presentdisclosure, are within the scope of the present disclosure.

INDUSTRIAL APPLICABILITY

The present disclosure provides a composition comprising catalase, apreparation method and use thereof and a method for killing tumor cells.The composition utilizes catalase to decompose hydrogen peroxide in thetumor to produce dissolved oxygen, thereby relieving the hypoxia intumor cells; solving the problems in the prior art involving theradiation resistance of hypoxic tumor cells; increasing the uniformityof the distribution of radioactive particles and the ability ofradiative rays to kill tumor cells; improving the problems in the priorart that the non-uniform distribution and poor coverage of radionuclidesthat has been placed in carrier particles in advance will occur afterbeing implanted into the lesion area, without affecting the surroundingnormal tissues. The composition has good prospects for tumor therapyapplications.

What is claimed is:
 1. A composition, comprising catalase, a solublealginate, and a radionuclide labeled on the catalase, wherein thesoluble alginate includes at least one selected from the groupconsisting of sodium alginate, potassium alginate, ammonium alginate andpropylene glycol alginate, and wherein in the composition aconcentration of the soluble alginate is 5 mg/mL-10 mg/mL.
 2. Thecomposition according to claim 1, wherein the soluble alginate is amixture of sodium alginate and potassium alginate.
 3. The compositionaccording to claim 1, wherein the radionuclide includes at least oneselected from the group consisting of iodine-131, iodine-125,phosphorus-32, yttrium-90, gallium-67, indium-111, thallium-201,palladium-203, bismuth-213, actinium-225, lutecium-177, rhenium-186,palladium-212 and rhenium-188.
 4. The composition according to claim 1,wherein the radionuclide is iodine-131 or iodine-125.
 5. The compositionaccording to claim 1, wherein the concentration of the catalase is 5mg/mL-10 mg/mL and the dose of the radionuclide is 200 Ci/mL-500 Ci/mL.6. The composition according to claim 1, wherein the composition furthercomprises an immune-stimulating agent.
 7. The composition according toclaim 6, wherein the immune-stimulating agent includes at least oneselected from the group consisting of CpG oligodeoxynucleotide which isa immunologic adjuvant, R837, TLR7 agonists, TLR8 agonists, NLRagonists, STING agonists, MPLA (Monophosphoryl Lipid A), LPS, PGNs,R848, G100, SD-101, MGN1703, CMP-001, FLA, polyU, poly(dT), CL307,CL264, CL097, CL075, MEDI9197, MEDI5083, hypoxanthine and MDP.
 8. Amethod for preparing a composition comprising catalase, comprisingmixing an alginate solution, a radionuclide and catalase.
 9. The methodaccording to claim 8, wherein a ratio of amount of the alginatesolution, catalase, and radionuclide is 5 mg-10 mg: 5 mg-10 mg: 200Ci-500 Ci.
 10. The method according to claim 8, wherein the method ofmixing includes mixing an alginate solution with a radionuclide-labeledcatalase.
 11. The method according to claim 10, wherein theradionuclide-labeled catalase is prepared by a method comprising mixingthe radionuclide with an oxidizing agent and a dispersion medium, andmixing the obtained mixture with the catalase.
 12. The method accordingto claim 10, wherein the radionuclide-labeled catalase is prepared by amethod comprising mixing the catalase with a chelating agent, mixing theobtained mixture with the radionuclide and a reducing agent, andpurifying.
 13. The composition according to claim 1 for use inpreparation of an anti-neoplastic agent.
 14. The composition accordingto claim 13, wherein the composition is used in combination with atleast one selected from the group consisting of an antibody againstcytotoxic T-lymphocyte-associated antigen-4, anti-PD-1 and anti-PD-L1.